Method for the fermentative production of L-valine and suitable microorganism

ABSTRACT

The invention relates to a method for producing L-valine and to a suitable microorganism. The inventive method is characterized by preferably enhancing the transaminase C activity of a  coryneform bacterium , especially  Corynebacterium glutamicum . The organisms so modified have a yield in L-valine which is 35.8% higher than that of non-modified organisms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the US national phase of PCT applicationPCT/DE2005/001465, filed 19 Aug. 2005, published 6 Apr. 2006 as WO2006/034667, and claiming the priority of German patent application102004046933.4 itself filed 28 Sep. 2004.

FIELD OF THE INVENTION

The invention relates to a method for the production of L-valine as wellas a suitable microorganism.

BACKGROUND OF THE INVENTION

The amino acid L-valine is used in human medicine, in the pharmaceuticalindustry, in the food industry as well as in pet food.

It is known that amino acids are produced from the fermentation ofstrains of coryneform bacteria, particularly corynebacterium glutamicum.Due to their great importance, the manufacturing processes arecontinually improved. Manufacturing improvements can relate tofermentation measures, such as agitation and supply with oxygen forexample, or to the composition of the nutrient solutions, such as theglucose concentration during fermentation, or the processing into theproduct form, for example through ion exchange chromatography or theintrinsic performance characteristics of the microorganism itself.

Methods of mutagenesis, selection, and mutant selection are used toimprove the performance characteristics of these microorganisms. Bydoing so, strains are obtained that are resistant to anti-metabolites orthat are auxotrophic for regulatory significant metabolites and produceL-amino acids. Such a corynebacterium strain is described, for examplein the U.S. Pat. No. 5,521,074, which strain is resistant to L-valineand sensitive to fluoropyruvic acid. Furthermore, it is described in EP0287123 that corynebacteria with resistance to mycophenolic acids can beused advantageously for L-valine production. From EP 0519113 A1 and U.S.Pat. No. 5,658,766 it is also known that the mutants with mutatedvalyl-tRNA synthetase in combination with further mutations can be usedfor L-valine production. In addition, WO 001996006926 A1 describes aprocess for the production of L-valine, wherein a microorganism is usedthat requires the vitamin lipoic acid for growth and has a defect in theATPase.

Recombinant DNA technology is additionally used for improving theintrinsic characteristics of L-amino acids-producing strains ofcorynebacterium. The documents EP 1155139B1 and EP 0356739 B1, forexample, describe that the enhancement of the expression of thebiosynthesis genes ilvBN, ilvC, and ilvD is advantageously used for theproduction of L-valine. Furthermore, it is known from EP 1155139 B1 thatfor L-valine production the weakening or elimination of the threoninedehydratase gene ilvA and/or of genes of the pantothenate synthesis canbe used.

OBJECTS OF THE INVENTION

It is the object of the invention to provide a method and a suitablemicroorganism for the improved fermentative production of L-valine.

It is a further object of the invention to provide a method for thefermentative production of L-valine using coryneform bacteria, in whichtransaminase C is modified and/or transaminase C expression is enhanced.

SUMMARY OF THE INVENTION

According to the invention a method is disclosed for the fermentativeproduction of L-valine wherein Transaminase C activity in amicroorganism is increased.

It was found that the coryneform bacteria produce L-valine in animproved manner after enhancement of the genes coding for transaminaseC. With the method according to the invention, it is now possible toproduce L-valine in a yield that is 35.8% higher than with a strain thatis not modified according to the invention.

Following the description of the invention.

According to the invention, the transaminase C activity is enhanced in amicroorganism that produces the amino acid L-valine.

The strains used preferably produce L-valine already before enhancementof the transaminase C gene.

As microorganism, a coryneform bacterium is preferably used.

Particularly preferred are corynebacterium glutamicum, corynebacteriumacetoglutamicum, corynebacterium thermoanimogenes, brevibacteriumflavum, brevibacterium lactofermentum, brevibacterium divaricatum.

Particularly suited strains of the category corynebacterium,particularly of the type corynebacterium glutaminum, are for example theknown wild-type strains

Corynebacterium glutaminum ATCC13032

Corynebacterium acetoglutamicum ATCC15806.

Corynebacterium acetoacidophilum ATCC13870

Corynebacterium thermoaminogenes FERN BP-1539

Brevibacterium flavum ATCC14067

Brevibacterium lactofermentum ATCC13869 and

Brevibacterium divaicatum ATCC14020 and

mutants, or strains produced from these and producing an excess ofL-amino acid.

The microorganisms that are the object of the present invention canproduce L-valine for example from glucose, saccharose, lactose,fructose, maltose, molasses, starches, cellulose, or from glycerin andethanol. They are representatives of coryneform bacteria, particularlythe type corynebacterium. From the category corynebacterium,particularly the type corynebacterium glutamicum should be mentioned,which is known among experts for its ability to produce L-amino acids.

In terms of the invention, the term “enhancement” is interpreted as theincrease of intracellular transaminase C activity, e.g. throughfollowing actions:

Increase of the gene expression through at least one step from the groupcomprising:

Modifications of the signal structures of the gene expression, such asthrough modification of the

repressor genes,

activator genes,

operators,

promoters;

attenuators,

ribosome binding sites,

start codon,

terminators

Introduction of a stronger promoter, such as a tac-promoter, or anIPTG-inducible promoter for example.

Increase of the gene copy count, e.g. by introduction of vectors likeplasmides, increase of the endogenous gene copy count, meaning theintroduction of further genes coding for transaminase C or allelesthereof into the chromosomal genome.

Furthermore, an enhancement of the transaminase C activity can beprovoked through the following measures:

Increase of the m-RNA-stability, for example through mutation ofterminal positions that control the termination of transcription.

For example, through stability of the m-RNA of the transaminase C genean improved product creation can be achieved in that the stability ispositively influenced by additional and/or modified sequences on the5′-end or the 3′-end of the gene. General examples for this are genesfrom bacillus subtilis (Microbiology 2001, 147:1331-41) or yeast (TrendsBiotechnol. 1994, 12:444-9).

Use of a gene or allele that codes for a corresponding enzyme withincreased activity.

The increase of the intracellular activity of one or more enzymes(proteins) within a microorganism, which enzymes are coded for by thecorresponding DNA, can be enhanced by the use of a strong promoter or agene, or as the case may be, by an allele that codes for a correspondingenzyme with increased activity, or overexpresses the corresponding gene(protein) and, as the case may be, combines these measures.

The introduction of a stronger promoter, such as the tac-promoter (Amannet al (Gene 1988 69:301-15)) for example, or promoters from the group ofpromoters described in Patek et al (Microbiology 1996 142:1297), ispreferred. Examples can be found in WO 96/15246 or in Boyd and Murphy(Journal of Bacteriology 170: 5949 (1998)), in Voskuil and Chambliss(Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer(Biotechnology and Bioengineering 58: 191 (1998)), in Pátek et al(Microbiology 142: 1297 (1996)), Knippers (“Molekulare Genetic[Molecular Genetics]”, 6^(th) edition, Georg Thieme publishing house,Stuttgart, Germany 1995), or also at Winnacker (“Gene und Klone [Genesand Clones]”, VCH publishing company, Weinheim, Germany, 1990).

The natural nucleotide sequence of the transaminase C gene is inevitablyknown as a result of the creation of the complete genome sequence of C.Glutamicum (Kalinowski et al, 2003, J. Biotechnol., 104:5-25; Ikeda M,and Nakagawa S. 2003 Appl. Microbiol. Biotechnol. 62:99-109), howeverwithout knowledge of the association of an open reading frame fortransaminase C. It is also known that c. Glutamicum (Leyval et al 2003.J. Biotechnol. 104:241-52) as well as for example E. coli havetransaminase C activity (Wang et al 1987, J. Bacteriol. 169:4228-4234).The open reading frame as subsequently described and identified in theexample, which codes for transaminase C, bears the number NCg12510 andhas SEQ ID NO: 1 which encodes Transaminase C from Coryneform Glutamicumhaving SEQ ID NO: 2, and is stored in the publicly accessible databaseof the “National Institute of Health” (ncbi.nlm.nih.gov), the identicalgene also being identified under Cg12599 in the publicly accessible “DNAData bank of Japan” (gib.genes.nig.ac.jp).

The transaminase-C-gene described by these numbers is preferably usedaccording to the invention. Furthermore, the allele of the transaminaseC gene can be used, which for example results from the degeneration ofthe genetic code or from functionally neutral sense mutations or fromthe deletion or insertion of nucleotides.

In order to achieve an enhancement, either the expression of thetransaminase C gene or the catalytic properties of the enzyme proteincan be increased, or as the case may be enhanced. The catalytic propertyof the enzyme protein can also be modified in regard to its substratespecificity. If necessary, both measures can be combined.

The enhancement of gene expression can take place through suitableculture management, or through genetic modifications (mutation) of thesignal structures of the gene expression. The signal structures of thegene expression are, for example, repressor genes, activator genes,operators, promoters, attenuators, ribosome binding sites, the startcodon, and terminators. The person skilled in the art can findinformation hereunto e.g. in the patent application WO 96/15246, at Boydand Murphy (J. Bacteriol. 1988.170: 5949), at Voskuil and Chambliss(Nucleic Acids. Res. 1998. 26: 3548), at Jensen and Hammer (Biotechnol.Bioeng. 1998 58: 191), at Patek et al. (Microbiology 1996. 142:1297),and in known textbooks for genetics and molecular biology, such as forexample the textbook by Knippers (“Molekulare Gentechnik [MolecularGenetics]”, 8^(th) edition, Georg Thieme publishing house, Stuttgart,Germany, 2001), or one by Winnacker (“Gene und Klone [Genes andClones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

Mutations resulting in a modification of the catalytic properties ofenzyme proteins, particularly in modified substrate specificity, areknown from the state of the art. As an example, the works of Yano et al1998 Proc Natl Acad Sci U S A. 95:5511-5, Oue S. et al J. Biol Chem.1999, 274:2344-9, and Onuffer et al Protein Sci. 1995 4:1750-7 should bementioned, in which the modification of the specificity of aspartateaminotransferases is revealed. Transitions, transversions, insertions,and deletions can be considered as mutations, as well as methods ofdirected evolution. Instructions for the production of such mutationsand proteins are part of the state of the art and can be gathered fromknown textbooks (R. Knippers “Molekulare Genetik [Molecular Genetics]”,8^(th) edition, 2001 Georg Thieme publishing house, Stuttgart, Germany),or review articles (N. Pokala 2001, J. Struct. Biol. 134:269-81; A.Tramontano-2004, Angew. Chem. [Applied Chemistry] Int. Bd Engl.43:3222-3; N. V. Dokholyan 2004, Proteins. 54:622-8; J. Pei 2003, Proc.Natl. Acad. Sci U S A. 100:11361-6; H. Lilie 2003, EMBO Rep. 4:346-51;R. Jaenicke Angew. Chem. [Applied Chemistry] Int. Ed. Engl. 42:140-2).

The expression of the genes or mutated genes preferably takes placeaccording to conventional methods of increasing the copy count throughthe integration into suitable plasmids. Plasmids that are replicated incoryneform bacteria are suitable. Numerous known plasmid vectors, suchas pz1 (Menkel et al, Applied and Environmental Microbiology (1989)64:549-554), pEKEx1 (Eikmanns et al, Gene 102:93-98 (1991)), or pHS2-1(Sonnen et al, Gene 107:69-74 (1991)) for example, are based on thecryptic plasmids pHM1519, pBL1 or bGA1. Other plasmid-vectors, as forexample those that are based on pCG4 (U.S. Pat. No. 4,489,160) or pNG2(Serwold-Davis et al, FEMS Microbiology Letters 66, 119-124 (1990)) orpAG1 (U.S. Pat. No. 5,158,891) can also be used (O. Kirchner 2003, J.Biotechnol. 104:287-99). Vectors with adjustable expression, such as forexample pEKEx2 (B. Eikmanns, 1991 Gene 102:93-8; O. Kirchner 2003, J.Biotechnol. 104:287-99), can be used as well. The gene can also beexpressed through integration in the chromosome in single copy (P.Vasicova 1999, J. Bacteriol. 181:6188-91), or multiple copies (D.Reinscheid 1994 Appl. Environ Microbiol 60:126-132).

The transformation of the desired strain with the vector in order toincrease the copy count takes place by conjugation or electrophorationof the desired strain of C. glutamicum, for example. The method ofconjugation is described by Schäfer et al (Applied and EnvironmentalMicrobiology (1994) 60:756-759), for example. Methods for transformationare described by Tauch et al (FEMS Microbiological Letters (1994)123:343-347) for example.

This way, the transaminase C gene or its allele can be expressed oroverexpressed in C. glutamicum.

Furthermore, it can be advantageous for the production of L-valine, inaddition to increasing the transaminase C activity, to enhance one ormore genes chosen from the group of

the ilvBN gene coding for acetohydroxyacid synthase,

the gene coding for isomer reductase,

the ilvD gene coding for dehydratase

particularly to overexpress, or to enhance or overexpress alleles ofthese genes, in particular

the ilvBN genes coding for feedback-resistant acetohydroxy acidsynthase,

in order to further increase the production of L-valine.

Furthermore, it can be advantageous for the production of L-valine, inaddition to increasing the transaminase C activity, to deactivate orreduce in their expression, or to mutate, one or more genes chosen fromthe group of

the panBCD genes coding for pantothenate synthesis,

the lipAB genes coding for lipoic acid synthesis,

the aceE, aceF, 1pD genes coding for pyruvate dehydrogenase,

the genes for the genes of the ATP synthase A subunit, ATP synthase Bsubunit, ATP synthase C subunit, ATP synthase alpha subunit, ATPsynthase gamma subunit, ATP synthase subunit, ATP synthase epsilonsubunit, ATP synthase delta subunit, in order to create-functionallyweakened gene products, so the production of L-valine can be increased.

The microorganisms produced according to the invention can be cultivatedcontinuously or discontinuously in a batch procedure (batch cultivation)or in the fed batch (feed procedure) or repeated fed batch procedure(repetitive feed procedure) for the purpose of valine production. Asummary of known cultivation procedures is described in the textbook ofChmiel (Bioprozesstechnik 1. Einfuehrung in die Bioverfahrenstechnik[Bioprocess Technology 1^(st) Introduction into Bio-procedureTechnology] (Gustav Fischer Verlag, Stuttgart, 1991)), or in thetextbook by Storhas (Bioreaktoren und periphere Einrichtungen[Bioreactors and Peripherals Devices] (Vieweg publishing house,Braunschweig/Wiesbaden (1994)).

The culture medium to be used must meet the requirements of therespective microorganisms. Descriptions of culture media of differentmicroorganisms are included in the handbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981).

Possible carbon dioxide sources include sugar and carbohydrates, such asglucose, saccharose, lactose, fructose, maltose, molasses, starch andcellulose, oils and fats, such as soy oil, sunflower seed oil, peanutoil and coconut oil, fatty acids, such as palmitic acid, stearic acidand linoleic acid, alcohols such as glycerin and ethanol, as well asorganic acids, such as acetic acid.

These substances can be used individually or as a mixture.

Possible nitrogen sources are organic, nitrogen-containing compoundslike peptones, yeast extract, meat extract, malt extract, corn steepliquor, soybean flour and urea, or inorganic compounds like ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium-carbonate, andammonium nitrate. The nitrogen sources can be used individually or as amixture.

As the phosphorus source, potassium dihydrogen phosphate or di-potassiumhydrogen phosphate, or the corresponding sodium-containing salts can beused.

Furthermore, the culture medium should include salts of metals, such asmagnesium sulfate, or ferric sulfate for example, which are necessaryfor growth. Lastly, essential growth substances, such as amino acids andvitamins can be used in addition to the above-mentioned substances. Theingredients mentioned above can be added to the culture in the form of aone-time mixture, or they can be added in a suitable manner duringcultivation.

In order to control the pH level of the culture, basic compounds likesodium hydroxide, potassium hydroxide, ammonia, or acid compounds likephosphoric acid, or sulfuric acid can be used in an appropriate manner.To control foam development, de-foaming agents, such as fatty acidpolyglycolester, can be used. In order to maintain the stability of theplasmids, selective substances, for example antibiotics, can be added tothe medium. In order to maintain aerobic conditions, oxygen oroxygen-containing gas mixtures, for example air, can be introduced intothe culture. The temperature of the culture is normally between 20° C.and 45° C., and preferably between 25° C. and 40° C. The culture iscultivated until a maximum of L-valine has formed. This goal is usuallyreached within 10 hours to 160 hours.

BRIEF DESCRIPTION OF THE DRAWING

The sole FIGURE in this case is a map of plasmid pEKEx2ATC, used for thetransformation of the Corynebacterium glutamicum strain 13032ΔpanBC tooverexpress Transaminase C from Corynebacterium glutamicum to catalyzethe biosynthesis of L-valine from L-alanine and ketoisovalerate.

EXAMPLES Example 1 Cloning of Transaminase C

With the aid of the PCR reaction, a DNA fragment comprising thetransaminase C gene having SEQ ID NO: 1, was amplified. The followingprimer were used:

orf2841-for: 5′-ATGGTA (GGTCT) CAAATGTCTCTTATGAAGCCAAGCACTAG-3′orf2841-rev (SEQ ID NO: 3): (SEQ ID NO: 4)5′-ATGGTA (GGTCT) CAGCGCTTTTTTTGATGAATTCTCCGATTTT G-3′

The primers listed were synthesized by MWG Biotech, and the PCR reactionwas carried out in accordance with standard protocols (Innis et al PCRProtocols. A. Guide to Methods and Applications. 1990. Academic Press).A DNA fragment of about 1.1 kb was amplified with the primers thatfragment codes for transaminase C. The primers comprise additionally theinterface of the restriction enzyme BsaI that are shown in the abovenucleotide sequences in brackets.

The amplified DNA fragment of about 1.1 kb was identified in the 0.8%agarose gel and was isolated from the gel with existing methods (QIAquikGel Extraction Kit, Quiagen, Hilden). The ligation of the fragment wascarried out with the SureCloning Kit (Amersham, UK) into the expressionvector pASK-IBA-3C (IBA, Goettingen). With this ligation approach, E.coli DH5 was transformed (Grant et al, 1990. Proceedings of the NationalAcademy of Science of the United States of America USA, 87:4645-4649).The selection of plasmid-containing strains was done by plating thetransformation mixture onto LB-plates containing 25 mg ofchloramphenicol per liter.

After plasmid isolation, the resulting plasmids were characterized byrestriction digest and gel electrophoresis analysis. The resultingplasmid was labeled as pASK-IBA-3Corf2841.

Example 2 Isolation of Transaminase C

E. coli EH5 with pASK-IBA-3Corf2841 was cultured at 30° C. in 100 ml LBwith 25 mg of chloramphenicol per liter to an optical density of 0.5.Then 0.01 ml of an anhydrotetracycline solution was added that comprised2 mg of anhydrotetracycline per milliliter of dimethylformamide. Theculture was incubated for 3 more hours at 30° C. Afterward, the cellswere harvested through centrifugation for 12 minutes at 4° C. and 5000revolutions per minute. Then the cell pellet was resuspended in washbuffer (100 mm trihydroxymethylaminomethane, 1 mM ethylenediaminetetraacetic acid, pH8) and transferred to an Eppendorf reactiontube. The cell disruption occurred at 0° C. with an ultrasounddisintegrator (Branson Sonifier W-250, Branson Sonic Power Company,Danbury, USA; acoustic irradiation time 10 min., pulse length 20%,acoustic irradiation intensity 2). After the ultrasound treatment, thecell debris was separated by centrifugation (30 min., 13000 Rpm, 4° C.)and the raw extract was obtained as supernatant.

For the isolation of the protein StrepTactin-Affinity Columns from themanufacturer IBA (IBA, Göttingen, Germany) were filled with 1 ml bedvolume StrepTactin-sepharose. After equilibration of the columns withwash buffer from the manufacturer IBA, 1 ml of the raw extract wasapplied to the sepharose. After the passage of the extract, the affinitycolumn was washed 5 times with 1 ml wash buffer. The elution of thetransaminase-C protein was carried out with elution buffer, comprising100 mM Tris, 1 mM EDTA, 2.5 mM desthiobiotin, pH 8. The elutionfractions were aliquoted, frozen at −20° C., and used directly in theenzyme test.

Example 3 Activity Determination of Transaminase C

The reaction batch of the enzyme test comprised in a total volume of 1ml: 0.2 ml 0.25 M Tris/HCl, pH 8, 0.005 ml transaminase-C protein, and0.1 ml 2.5 mM pyridoxal phosphate, as well as 0.1 ml 40 mMketoisocaproate and 0.1 ml 0.5 M L-alanine, or 0.1 ml 40 mMketoisovalerate and 0.1 ml 0.5 M L-alanine, or 0.1 ml 40 mMketoisocaproate and 0.1 ml 0.5 M L-glutamine, or 0.1 ml 40 mMketoisocaproate and 0.1 ml 0.5 M L-alanine without transaminase-Cprotein. The enzyme test was carried out at 30° C. in a thermocycler5436 from the company Eppendorf (Hamburg). The reaction was started byadding the protein. By adding 30 μl of a stop reagent (6.7%) (v/v)perchloric acid (70%), 40% (v/v) ethanol (95% (in water)) to each 50 μlof the test batch, the enzyme test was stopped. In order to prepare thesamples for the verification of the formed aminoacids via reversed phaseHPLC, 20 μl of a neutralizing buffer (20 mM Tris, 2.3 M dipotassiumcarbonate, pH 8) was added. The deposit precipitated as a result of theneutralization of perchloric acid was centrifuged off (13000 Rpm, 10min) and the supernatant was used in different dilutions for thequantification via HPLC. This was carried out following automaticderivatization with o-phthaldialdehyde, as described (Hara et al 1985,Analytica Chimica Acta 172:167-173). As table 1 shows, the isolatedprotein having SEQ ID NO: 2 catalyses the L-alanine dependent aminationfrom ketoisovalerate into L-valine.

TABLE 1 Amino- Amino- Spec. Protein donator acceptor Product ActivityTransaminase C L-Alanine Ketoiso- L- 0.9 caproate Leucine Transaminase CL-Alanine Ketoiso- L-Valine 18.2 valerate Transaminase C L-AlanineKethometyl- L-Iso- 3.7 valerate leucine Transaminase C L- Kethoiso-L-Valine 0.1 Glutamine valerate Control L-Alanine Ketoiso- L-Valine 0.0valerateThe specific activity (spec. activity) is shown in micromol of productper minute and milligram transaminase C protein.

Example 4 Overexpression of Transaminase C

With the aid of the PCR reaction, a DNA-fragment comprising theTransaminase C gene was amplified. The following primers were used:

Trans_c_for: 5′-CGGGATCCAAGGAGATATAGATATGTCTCTTATGAAGCCAAGCA-3′Trans_c_rev (SEQ ID NO: 5): (SEQ ID NO: 6)5′-CGGGATCCCTATTTTTTGATGAATTCTCC-3′

The primers listed were synthesized by MWG Biotech, and the PCR reactionwas carried out according to standard protocols (Innis et al PCRProtocols. A Guide to Methods and Applications. 1990. Academic Press.) ADNA fragment of approximately 1.1 kb was amplified with the primers thatfragment codes transaminase C. The primers comprise additionally theinterface of the restriction enzyme BamHI.

The amplified DNA-fragment of approximately 1.1 kb was identified in0.8% agarose gel and isolated from the gel with existing methods(QIAquik Gel Extraction Kit, Quiagen, Hilden). The ligation of thefragment was carried out with the SureCloning Kit (Amersham, UK) intothe expression vector pEKEx2 (Bikmanns et al 1991, Gene 102:93-8). Withthe ligation batch, E. coli DH5 was transformed (Grant et al, 1990.Proceedings of the National of Sciences of the United States of AmericaUSA, 87:4645-4649). The selection of plasmid-comprising strains tookplace by plating the transformation batch to LB plates with 25 mg perliter of kanamycin.

After the plasmid isolation, the resulting plasmids were characterizedby restriction digest and gel electrophoresis analysis. The resultingplasmid was labeled pEKEx2ATC.

The plasmid pEKEx2ATC, as well as the starting plasmid pEKEx2 were usedfor the transformation of the 13032ΔpanBC strain to kanamycinresistance. The strain is described in EP1155139B1, and thetransformation technique in Kirchner et al J Biotechnol. 2003,104:287-99.

Example 5 L-Valine Formation

The 13032ΔpanBC pEKEx2ATC strain as well as the 13032ΔpanBC PEKEx2control strain were cultivated in the medium CGIII (Menkel et al 1989,Appl. Environ. Microbiol. 55:684-8) at 30° C. The medium CGIII wasinoculated with this at an optical density of 1. The medium CG12comprises per liter: 20 g (NH₄)₂SO₄, 5 g uric acid, 1 g KH₂PO₄, 1 gK₂HPO₄, 0.25 g Mg₂O₄.7 H₂O, 42 g 3-morpholinopropanesulfonic acid, 10 mgCaCl₂, 10 mg FeSO₄.7 H₂O, 10 mg MnSO₄. H₂O, 1 mg ZnSO₄.7 H₂O, 0.2 mgCuSo₄, 0.02 mg NiCl₂.6 H₂O, 0.2 mg biotin, 40 g glucose, and 0.03 mgprotokatechuic acid. The culture was incubated at 30° C. and 170revolutions per minute, and after 48 hours the L-valine accumulation inthe medium was determined with HPLC. This was carried out witho-phthaldialdehyde, as described (Hara et al 1985, Analytica ChimicaActa 172:167-173). The particular L-valine concentrations are shown intable 2.

TABLE 2 Strain L-valine 13032ΔpanBC 11.0 mM pEKEx2ATC 13032ΔpanBC  8.1mM pEKEx2

1. A method for increasing fermentative production of L-valine in aculture medium containing an L-valine-producing Coryneform bacterium,which comprises the step of transforming the L-valine-producingCoryneform bacterium by introducing therein a vector comprising at leastone polynucleotide coding for transaminase C having SEQ ID NO: 2 fromCorynebacterium glutamicum to obtain a recombinant L-valine-producingCoryneform bacterium having increased transaminase C activity over thatof the corresponding wild-type L-valine-producing Coryneform bacteriumthrough an increase in transaminase C gene expression, wherein theincreased transaminase C gene expression of the transformedL-valine-producing Coryneform bacterium over the corresponding wild-typeL-valine-producing Coryneform bacterium results from an exogenousincrease in the gene copy count of polynucleotides coding fortransaminase C from Corynebacterium glutamicum, and also fromdeactivation of pantothenate synthesis B (panB) and pantothenatesynthesis C (panC) genes.
 2. The method according to claim 1, wherein aplasmid is used as a vector.
 3. The method according to claim 2, whereinat least one component of the vector is a plasmid which is selected fromthe group consisting of pZ1, pEKEx1, pHS2-1, pHM1519, pBL1, pGA1, pCG4,pNG2, pAG1, and pEkEx2.
 4. The method according claim 1, wherein thewild-type L-valine-producing Coryneform bacterium is a Corynebacteriumselected from the group consisting of Corynebacterium glutamicum,Corynebacterium acetoglutamicum, Corynebacterium thermoanimogenesis,Brevibacterium flavum, Brevibacterium lactofermentum, and Brevibacteriumdivaicatum.
 5. The method according to claim 4, wherein the wild-typeL-valine-producing Coryneform bacterium is selected from the groupconsisting of microorganisms Corynebacterium glutamicum ATCC13032,Corynebacterium acetoglutamicum ATCC15806, Corynebacteriumacetoacidophilum ATCC 13870, Corynebacterium thermoaminogenes PERMBP-1539, Brevibacterium flavum ATCC14067, Brevibacterium lactofermentumATCC13869, and Brevibacterium divaricatum ATCC14020.
 6. The methodaccording to claim 1, wherein a Coryneform bacterium is used thatcomprises a Transaminase C gene having SEQ ID NO: 1 from Corynebacteriumglutamicum with the gene identification number NCgl2510 of the databaseof the “National Institute of Health”.
 7. The method according to claim2 wherein the L-valine-producing Coryneform bacterium is 13032ΔpanBCtransformed by introducing as the vector the plasmid pEKEx2ATC.